Automated liquid manufacturing system

ABSTRACT

A method for continuously preparing a medium formulation mixes a diluent with a plurality of chemically incompatible concentrate solutions in such a manner that none of the ingredients of the concentrate solutions chemically react in an adverse manner. The method utilizes a static mixing chamber to add the concentrate solutions to the diluent stream sufficiently in advance of one another so that adverse chemical reactions do not occur. The method also adjusts a pH level of the diluent prior to adding any of the concentrate solutions to the diluent.

This patent application is a divisional of U.S. patent application Ser.No. 08/857,496, filed on May 16, 1997, now issued as U.S. Pat. No.6,004,025.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the field of cell culture mediumformulations, and more specifically, to methods for continuouslypreparing cell culture medium formulations and buffered salt solutionsfrom selected subgroups of medium concentrates.

2. Related Art

Cell culture medium formulation provide nutrients necessary to maintainand grow cells in a controlled, artificial and in vitro environment.Characteristics and compositions of the cell culture mediums varydepending on the particular cellular requirements. Important parametersinclude osmolarity, pH, and nutrient formulations.

Medium formulations have been used to grow a number of cell typesincluding animal, plant and bacterial cells. Cells grown in culturemedium catabolize available nutrients and produce useful biologicalsubstances such as monoclonal antibodies, hormones, growth factors andthe like. Such products have therapeutic applications and, with theadvent of recombinant DNA technology, cells can be engineered to producelarge quantities of these products. Thus, the ability to grow cells invitro is not only important for the study of cell physiology, it isnecessary for the production of useful substances which may nototherwise be obtained by cost-effective means.

Cell culture medium formulations have been well documented in theliterature and a number of medium are commercially available. Typicalnutrients in cell culture medium formulations include amino acids,salts, vitamins, trace metals, sugars, lipids and nucleic acids. Often,particularly in complex medium formulations, stability problems resultin toxic products and/or lower effective concentrations of requirednutrients, thereby limiting the functional life-span of the culturemedium. For instance, glutamine is a constituent of almost all mediumformulations that are used in the culturing of mammalian cells in vitro.Glutamine decomposes spontaneously into pyrrolidone carboxylic acid andammonia. The rate of degradation can be influenced by pH and ionicconditions but in cell culture medium, formation of these breakdownproducts cannot be avoided (Tritsch et al., Exp. Cell Research,28:360-364(1962)).

Wang et al. (In Vitro, 14:(8):715-722 (1978)) have shown thatphotoproducts such as hydrogen peroxide, which are lethal to cells, areproduced in Dulbecco's Modified Eagle's Medium (DMEM). Riboflavin andtryptophan or tyrosine are components necessary for formation ofhydrogen peroxide during light exposure. Because most mammalian culturemedium formulations contain riboflavin, tyrosine and tryptophan, toxicphotoproducts are likely produced in most cell culture mediums.

To avoid these problems, researchers make medium formulations on an “asneeded” basis, and avoid long term storage of the culture medium.Commercially available medium formulations, typically in dry powderform, serve as a convenient alternative to making the mediumformulations from scratch, i.e., adding each nutrient individually, andalso avoids some of the stability problems associated with liquid mediumformulations. However, only a limited number of commercial culturemedium formulations are available, except for those custom formulationssupplied by the manufacturer.

Although dry powder medium formulations may increase the shelf-life ofsome medium formulations, there are a number of problems associated withdry powdered medium formulations, especially in large scale application.Production of large volumes requires storage facilities for the drypowder, not to mention the specialized kitchens necessary to mix andweigh the nutrient components. Due to the corrosive nature of dry powdermedium ingredients, mixing tanks must be periodically replaced.

There exists a need to lower the cost of production of biologicalsubstances. Efficient and cost effective methods to stabilize liquidcell culture medium formulations as well as the development ofconvenient methods to produce 1×medium formulations would be animportant development in the field of cell culture medium technology.

One such development in the field of cell culture medium formulations isthe development of liquid medium concentrates as is disclosed in U.S.Pat. No. 5,474,931 issued to DiSorbo et al. on Dec. 12, 1995(“DiSorbo”). DiSorbo discloses a method of subgrouping mediumformulations into stable, compatible components that can be solubilizedat high concentrations (10× to 100×). Concentrated culture mediumformulations (2-10×) or 1×cell culture medium formulations can beprepared by mixing a sufficient amount of the concentrated subgroupsolutions with each other and with a sufficient amount of a diluent(water, buffer, etc.).

Escalating demand for large volumes of nutrient medium and buffered saltsolutions and increasing pressure to minimize batch-associated costs,such as sterile filtration and quality release testing, has driven arequirement for increased production batch sizes of liquid medium. As aresult, stainless steel formulation tanks of 5000-10,000 liters forpreparation of large batches of liquid medium or buffered salt solutionshave become relatively common. However, scale-up manufacture of thesefluids in this manner presents challenges regarding product quality andeconomy.

What is needed is a system and method for providing continuous, onlinepreparation of large volumes of biological fluids (e.g., liquid medium,buffered salt solutions, etc.) within a highly controlled manufacturingsystem.

SUMMARY OF THE INVENTION

The present invention is a system and method for continuous, onlinepreparation of cell culture medium formulations from selected subgroupsof medium concentrates. In particular, a computer controlled systemcontrols the flow of a diluent and one or more concentrated solutionsinto a static mixing chamber wherein the diluent and the concentratedsolutions are mixed to form the cell culture medium formulations.

The present invention is able to formulate a cell culture medium fromconcentrated solution subgroups including an acid soluble concentratesolution subgroup, a group I salts solution concentrate subgroup, agroup II salts solution concentrate subgroup, and a base solublesolution concentrate subgroup. Furthermore, the present invention isable to adjust the pH of the cell culture medium using either an acidsolution or a base (caustic) solution.

In particular, the present invention is able to mix the concentratedsolution subgroups with the diluent in a manner such that theingredients of the concentrated solution subgroups do not adverselyreact chemically with one another.

One feature of the present invention is the preparation of largequantities of 1×cell culture medium (100,000 liters or more) whilerequiring only one quality control test. By increasing the size of the“batch,” the present invention reduces the per liter cost of cellculture medium.

Another feature of the present invention is the increased consistency inthe 1×cell culture medium. Statistical analyses have demonstrated thatthe present invention is able to provide 1×cell culture medium withhomogeneity within batches of ±2.0%. Furthermore, the present inventionprovides improved precision between production runs of 1×cell culturemedium manufactured from identical concentrate solutions of ±3.0%.

Still another feature of the present invention is a clean in place (CIP)and a steam in place (SIP) system which allows various components of thepresent invention to be sanitized and sterilized according to currentgood manufacturing practices (cGMP).

Further features and advantages of the present invention, as well as thestructure and operation of various embodiments of the present invention,are described in detail below with reference to the accompanyingdrawings.

BRIEF DESCRIPTION OF THE FIGURES

The present invention is described with reference to the accompanyingdrawings. In the drawings, like reference numbers indicate identical orfunctionally similar elements. Additionally, the left-most digit(s) of areference number identifies the drawing in which the reference numberfirst appears.

FIG. 1 illustrates an automated liquid manufacturing system (ALMS)according to the present invention.

FIG. 2 illustrates a diluent system according to a preferred embodimentof the present invention.

FIG. 3 illustrates a medium mixing system according to a preferredembodiment of the present invention.

FIG. 4 illustrates a medium surge vessel according to one embodiment ofthe present invention.

FIG. 5 illustrates a pre-filtration system and a sterile filtrationsystem according to a preferred embodiment of the present invention.

FIGS. 6A and 6B, respectively, illustrate a front view and a right sideview of a medium mixing chamber according to a preferred embodiment ofthe present invention.

FIG. 7 illustrates an isometric view of a portion of the medium mixingchamber according to a preferred embodiment of the present invention.

FIG. 8 illustrates an example of a computer control system useful forcontrolling the operation of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the description that follows, a number of terms conventionally usedin the field of cell culture medium are utilized extensively. In orderto provide a clear and consistent understanding of the specification andclaims, and the scope to be given such terms, the following definitionsare provided.

Ingredients. The term “ingredients” refers to any compound, whether ofchemical or biological origin, that can be used in cell culture mediumto maintain or promote the growth or proliferation of cells. The terms“component,” “nutrient,” and “ingredient” can be used interchangeablyand are all meant to refer to such compounds. Typical ingredients thatare used in cell culture medium formulations include amino acids, salts,metals, sugars, lipids, nucleic acids, hormones, vitamins, fatty acids,proteins and the like. Other ingredients that promote or maintain growthof cells in vitro can be selected by those of skill in the art, inaccordance with the particular need.

Cell Culture. By “cell culture” is meant cells or tissues that aremaintained, cultured or grown in an artificial, in vitro environment.

Culture Vessel. Glass, plastic or metal containers of various sizes thatcan provide an aseptic environment for growing cells are termed “culturevessels.”

Cell Culture Medium. The phrases “cell culture medium” or “culturemedium” or “medium formulation” or “cell culture medium formulation”refer to a nutritive solution for culturing or growing cells. Theingredients that comprise such medium formulations may vary depending onthe type of cell to be cultured. In addition to nutrient composition,osmolarity and pH are considered important parameters of culture mediumformulations.

Compatible Ingredients. Each ingredient used in cell culture mediumformulations has unique physical and chemical characteristics. By“compatible ingredients” is meant those medium nutrients which can bemaintained in solution and form a “stable” combination. A solutioncontaining “compatible ingredients” is said to be “stable” when theingredients do not degrade or decompose substantially into toxiccompounds, or do not degrade or decompose substantially into compoundsthat can not be utilized or catabolized by the cell culture. Ingredientsare also considered “stable” if degradation can not be detected or whendegradation occurs at a slower rate when compared to decomposition ofthe same ingredient in a 1×cell culture medium formulation. Glutamine,for example, in 1×medium formulations, is known to degrade intopyrrolidone carboxylic acid and ammonia. Glutamine in combination withdivalent cations are considered “compatible ingredients” since little orno decomposition can be detected over time.

Compatibility of medium ingredients, in addition to stabilitymeasurements, are also determined by the “solubility” of the ingredientsin solution. The term “solubility” or “soluble” refers to the ability ofan ingredient to form a solution with other ingredients. Ingredients arethus compatible if they can be maintained in solution without forming ameasurable or detectable precipitate. Thus, the term “compatibleingredients” as used herein refers to the combination of particularculture medium ingredients which, when mixed in solution either asconcentrated or 1×medium formulations, are “stable” and “soluble.”

1×Formulation. A cell culture medium is composed of a number ofingredients and these ingredients vary from medium to medium. A“1×formulation” or “1×medium formulation” is meant to refer to anyaqueous solution that contains some or all ingredients found in a cellculture medium. The “1×formulation” can refer to, for example, the cellculture medium or to any subgroup of ingredients for that medium. Theconcentration of an ingredient in a 1×solution is about the same as theconcentration of that ingredient found in the cell culture formulationused for maintaining or growing cells. Cell culture medium formulationsused to grow cells are 1×formulation by definition. When a number ofingredients are present (as in a subgroup of compatible ingredients),each ingredient in a 1×formulation has a concentration about equal tothe concentration of those ingredients in a cell culture medium. Forexample, RPMI 1640 culture medium contains, among other ingredients, 0.2g/l L-arginine, 0.05 g/l L-asparagine, and 0.02 g/l L-aspartic acid. A“1×formulation” of these amino acids, which are compatible ingredientsaccording to the present invention, contains about the sameconcentrations of these ingredients in solution. Thus, when referring toa “1×formulation,” it is intended that each ingredient in solution hasthe same or about the same concentration as that found in the cellculture medium being described. The concentrations of medium ingredientsin a 1×formulation are well known to those of ordinary skill in the art,See Methods For Preparation of Media, Supplements and Substrate ForSerum-Free Animal Cell Culture, Allen R. Liss, N.Y. (1984), which isincorporated by reference herein in its entirety. The osmolarity and/orpH, however, may differ in a 1×formulation compared to the culturemedium, particularly when fewer ingredients are contained by the1×formulation.

10×Formulation. A “10×formulation” refers to a solution wherein eachingredient in that solution is about 10 times more concentrated than thesame ingredient in the cell culture medium formulation. RPMI 1640medium, for example, contains, among other things, 0.3 g/l L-glutamine.By definition, a “10×formulation” contains about 3.0 g/l glutamine. A“10×formulation” may contain a number of additional ingredients at aconcentration about 10 times that found in the 1×culture medium. As willbe apparent, “25×formulation,” “50×formulation” and “100×formulation”designate solutions that contain ingredients at about 25, 50 or 100 foldconcentrations, respectively, as compared to a 1×cell culture medium.Again, the osmolarity and pH of the medium formulation and concentratedformulation may vary.

Automated Liquid Manufacturing System

According to the present invention, an automated liquid manufacturingsystem (ALMS) continuously prepares medium products (e.g., cell culturemedium, buffered salt solutions, salt solutions, buffers, etc.) havingvarious formulations (e.g., 1-10×) by mixing one or more concentratesolution subgroups together with a diluent (e.g. water, buffer, etc.).The amount of concentrated solution and amount of diluent needed mayvary depending on the concentration of each subgroup, the number ofsubgroups, and the desired concentration of the final medium product.One of ordinary skill in the art can easily determined a sufficientvolume of a diluent and a sufficient volume of the concentratedsolutions to prepare the desired medium product.

The pH of the desired medium product may also be adjusted by theaddition of acid or base. The medium product, however, may not requireany adjustment, especially if the pH of the medium product as preparedis within the desired pH range. Osmolarity of the medium product canalso be adjusted after mixing the concentrated solutions with thediluent. Typically, the desired osmolarity may be predetermined andadjustments in the salt concentration of the concentrated solutions maybe made to prepare a final medium product with the desired osmolarity.

The present invention also provides for on-line sanitization andsterilization in place as required by current good manufacturingpractices (cGMP). The sanitization operation is commonly referred to as“clean in place,” and sterilization operation is commonly referred to as“steam in place.” These operations are discussed in further detailbelow.

According to the present invention, sufficient amounts of eachconcentrate solution subgroup are continuously admixed with sufficientamounts of a diluent in a mixing chamber, while the resulting mediumproduct is continuously removed. The following describes various aspectsof the present invention and the manner in which they accomplish thecontinuous preparation of medium product.

FIG. 1 illustrates a system level block diagram of an automated liquidmanufacturing system (ALMS) 100 according to the present invention. ALMS100 includes a concentrate system 110, a diluent system 120, a mediummixing system 130, a medium surge vessel 140, a prefiltration system150, a sterile filtration system 160 and a fill system 170. Sterilefiltration system 160 and fill system 170 operate in a clean area 180.In addition to the above-mentioned system components, a preferredembodiment of the present invention includes a waste disposal system190. Each of these components of ALMS 100 will be discussed in furtherdetail below.

A preferred embodiment of the present invention is controlled by acomputer control system 105. For ease of illustration, connectionsbetween computer control system 105 and the various components of ALMS100 have not been shown. Needless to say, each of the components of ALMS100 has some subcomponent, be it a valve, a pump, a sensor, etc., thatis connected to computer control system 105 and used to control theoperation of ALMS 100 as would be apparent. Computer control system 105is described in further detail below.

Concentrate System

Concentrate system 110 provides one or more concentrate solutions 115 toALMS 100. Specifically, concentrate system 110 provides concentratesolutions 115 to medium mixing system 130. Concentrate system 110 mayperform this task in a variety of ways. In one embodiment of the presentinvention, concentrate system may provide concentrate solutions 115 in amanner similar to that described in commonly owned U.S. Pat. No.5,474,931 issued to DiSorbo et al. on Dec. 12, 1995, which isincorporated herein by reference as if reproduced below in its entirety.DiSorbo discloses a method for producing liquid medium concentrates incompatible subgroups. According to this embodiment of the presentinvention, concentrate solutions 115 are chemically stable50×formulations of liquid medium concentrates.

These subgroups include the following: an acid soluble concentratesolution subgroup, a group I salts concentrate solution subgroup, agroup II concentrate solution subgroup, and a base soluble concentratesolution subgroup. In addition, sodium hydroxide may be prepared as aconcentrate solution subgroup although this is not necessary. The acidsoluble concentrate solution subgroup referred to herein is essentiallyequivalent to the acid-soluble subgroup referred to in DiSorbo; thegroup I salts concentrate solution subgroup referred to herein isessentially equivalent to the glutamine-containing subgroup referred toin DiSorbo; the group II salts concentrate solution subgroup referred toherein is essentially equivalent to the weak acid-base soluble subgroupreferred to in DiSorbo; and the base soluble concentrate solutionsubgroup referred to herein is essentially equivalent to thealkali-soluble subgroup referred to in DiSorbo. The remaining subgroupsreferred to in DiSorbo are treated as reserve concentrate solutions forpurposes of the present invention.

In this embodiment, the subgroups are formulated and “kited” accordingto published procedures as would be apparent. After being preparedaccording to these procedures the subgroups are stored in intermediatestorage vessels for use by ALMS 100.

In another embodiment of the present invention, concentrate system 110provides preformulated and prepackaged concentrate solutions 115. Theseconcentrate solutions 115 are purchased from a manufacturer of suchconcentrate solutions such as are available from Life Technologies,Incorporated, 3175 Staley Road, Grand Island, N.Y., 716/774-6700. Inaddition, concentrated subgroups for buffered salts can be obtained fromLife Technologies as acid soluble concentrate solution subgroups andbase soluble concentrate solution subgroups. This embodiment permits amanufacturer of medium products to purchase concentrate solutions 115without itself having the facilities to manufacture or produce suchconcentrate solutions 115.

In yet another embodiment of the present invention, concentrate system110 provides an on-line concentrate solution 115 as a part of acontinuous manufacturing process in which concentrate solutions 115 areproduced directly from raw materials and passed directly to ALMS 100without an intermediate storage device such as that described inDiSorbo.

As would be apparent to one skilled in the art, other types ofconcentrate solutions 115 are available other than the subgroupsdescribed above. Furthermore, other means for providing concentratesolution 115 to ALMS 100 may be available as would also be apparent.

Diluent System

Diluent system 120 provides a diluent 125 to ALMS 100. In particular,diluent system 120 provides diluent 125 to medium mixing system 130.Diluent 125 may be any solution or liquid that may be used to diluteconcentrate solutions 115. Such diluents include water, buffers, saltsolutions, etc. In a preferred embodiment of the present invention,diluent 125 is water, most preferably, water for injection. However, anydiluent 125 may be used in ALMS 100 that appropriately dilutesconcentrate solutions 115 according to the particular needs of themedium product manufacturer.

A preferred embodiment of diluent system 120 is illustrated in FIG. 2.In this embodiment of the present invention, diluent system 120 includesan ambient water for injection (WFI) tank 210, a hot WFI tank 220, acontrol valve 215, a control valve 225, and a WFI break tank 230. WFIbreak tank 230 includes a level indicator 250 and a spray ball 240.

The purpose of WFI break tank 230 is to provide an atmospheric breakbetween the plant water system and ALMS 100 as required by current goodmanufacturing practices (cGMP). In addition, WFI break tank 230 assuresremoval of entrained air from ambient WFI tank 210 and hot WFI tank 220prior to their introduction to ALMS 100.

In one embodiment of the present invention, ambient WFI tank 210 is nota tank. Rather, ambient WFI tank 210 is directly connected to theplant's water system. In other embodiments of the present invention,ambient WFI tank 210 may actually be a tank. This may be the case, forexample, when a diluent 125 other than water is used, or when aparticular type of water is required (e.g. deionized, distilled,sterile, etc.). Hot WFI tank 220 provides hot water to ALMS 100 during aclean-in-place (CIP) operation which is discussed in further detailbelow.

Valve 215 and valve 225 control the flow of ambient water from ambientWFI tank 210 and hot water from hot WFI tank 220, respectively, to WFIbreak tank 230. In a preferred embodiment of the present invention, WFIbreak tank 230 provides ambient water as diluent 125 to ALMS 100.

Level indicator 250 monitors a level of diluent 125 in WFI break tank230. Level indicator 250 is monitored by computer control system 105 tomaintain an appropriate level of diluent 125 in WFI break tank 230.

Spray ball 240 is a part of the CIP operation which is discussed infurther detail below. Spray ball 240 provides a mechanism for cleaningthe inside of WFI break tank 230 during the CIP operation.

Medium Mixing System

Medium mixing system 130 is shown in further detail in FIG. 3. Mediummixing system 130 includes a static mixing chamber 310, a diluent inputpump 320, a diluent flow indicator 325, a CIP divert valve 330, a seriesof concentrate solution pumps 340 (shown as concentrate solution pumps340A-H), a first pH sensor 361, a second pH sensor 362, a conductivitysensor 363, a UV absorbance sensor 364, an output flow indicator 365, adiverter valve 370, and a back flow preventer valve 375. Each of theseelements of medium mixing system 130 is described in further detailbelow.

Medium mixing system 130 receives diluent 125 and one or moreconcentrate solutions 115 and mixes them in mixing chamber 310. Mediummixing system 130 accomplishes this in a manner such that none of theingredients of concentrate solutions 115 adversely chemically react withone another or with diluent 125. By “adversely chemically react” it ismeant that the ingredients react 1) to form an irreversible precipitate;2) to cause degradation in one or more components of the concentratesolutions; 3) to cause certain components to become inactivated; or 4)to cause any other condition that would result in an unacceptable mediumproduct 135.

Diluent input pump 320 controls the flow of diluent 125 into staticmixing chamber 310. This flow is measured by diluent flow indicator 325.Diluent flow indicator 325 permits computer control system 105 tomonitor the flow of diluent 125 and thereby, control diluent input pump320. Back flow preventer valve 375 prevents diluent 125 from flowingbackwards from static mixing chamber

Based on the flow of diluent 125 into static mixing chamber 310,computer control system 105 controls the flows of concentrate solutions115 (shown as concentrate solutions 115A-H) into static mixing chamber310 via concentrate solution pumps 340 (shown as concentrate solutionpumps 340A-H). The flow of each of concentrate solutions 115A-H iscontrolled to be proportional to the flow of diluent 125 into staticmixing chamber 310 according to a formulation of a desired mediumproduct.

Sensors 361, 362, 363, 364 and 365 monitor a medium product 135 outputfrom static mixing chamber 310 to ensure that particular parametersassociated with medium product 135 are within acceptable levelsassociated with the desired medium product. These sensors are coupled tocomputer control system 105 which monitors these parameters of mediumproduct 135 to ensure that proper mixing of concentrate solutions 115A-Hand diluent 125 is being accomplished.

If the medium product is within the acceptance levels, medium product135 passes to medium surge vessel 140. If not, computer control system105 diverts medium product 135 to waste disposal system 190 via divertervalve 370. This allows medium mixing system 130 to guarantee anacceptable medium product 135. For example, when ALMS 100 starts uppreparation of a particular medium product 135, the initial output ofstatic mixing chamber 310 may not be within the acceptance levels forthe particular medium product. Thus, this portion of the output isdiverted to waste disposal system 190. When the output of static mixingchamber 310 enters into the acceptable levels (i.e., the operationreaches a “steady state”), the output from static mixing chamber 310 ispassed to medium surge vessel 140.

In a preferred embodiment of the present invention, first pH sensor 361and second pH sensor 362 are placed in close proximity to each other andas close to static mixing chamber 310 as possible, and prior to sensors363, 364 to ensure that the proper pH levels of medium product 135 isbeing achieved.

Conductivity sensor 363 measures the ionic character of medium product135. In particular, conductivity sensor 363 measures the resistivity ofthe flow of medium product 135. Conductivity sensor 363 is useful fordetermining the quality of medium product 135, especially for saltsolutions.

UV absorbance sensor 364 measures an amount of ultraviolet light thatpasses through the flow of medium product 135. UV absorbance sensor 364is useful for detecting the presence of precipitates within mediumproduct 135. UV absorbance sensor 364 can also be used to measure aconcentration of a particular component as an on-line measurement ofconcentrate addition and mixing quality.

As would be apparent to one skilled in the art, other types of sensorsmay be implemented in medium mixing system 130 to measure other levelsof other parameters associated with medium product 135.

In a preferred embodiment of the present invention, concentrate solutionpumps 340A-H are extremely precise variable speed pumps. In particular,concentrate solution pumps 340A-F are capable of delivering 0 to 3liters of fluid per minute with ±1.0% or better accuracy. Concentratesolution pumps 340G-H are capable of delivering 0 to 3.5 liters of fluidper minute with ±1.0% accuracy. A preferred embodiment of the presentinvention uses pumps which are manufactured by IVEK, North Springfield,Vt.

In a preferred embodiment of the present invention, concentrate solution115A and concentrate solution 115B are reserved for providing an acidsolution and a base solution, respectively, to static mixing chamber310. Hence, referring to these as “concentrate solutions” may beconsidered a misnomer. However, as would be apparent, solutions,liquids, etc., other that “concentrate solutions” may be introduced inthis manner to static mixing chamber 310 as would be apparent.

In this preferred embodiment of the present invention, acid solution115A and caustic solution 115B adjust a pH level of diluent 125according to specifications required by the production of medium product135. The addition of either acid solution 115A or caustic solution 115Bto diluent 125 is done first so that the proper pH level of diluent 125can be achieved prior to the addition of other concentrate solutions115C-H.

As shown in FIG. 3, diluent 125 enters static mixing chamber 310 andbegins “mixing” sufficiently prior to the addition of any concentratesolutions 115A-H. This ensures that static mixing chamber 310 canprovide a “turbulent diluent stream” from diluent 125 to enhance theoverall mixing process between diluent steam 125 and concentratesolution 115A-H. The turbulent diluent stream is produced from diluent125 by being forced past a series of baffles within static mixingchamber 310 as is well understood by those in the art. Also, theintroduction of a last concentrate solution 115H occurs sufficientlyprior to the end of static mixing chamber 310 so that last concentratesolution 115H can be sufficiently mixed in turbulent diluent stream. Asdiscussed above, the output of static mixing chamber 310 is mediumproduct 135.

As shown in FIG. 3, static mixing chamber 310 includes a series ofinjection ports 315 (shown as injection ports 315A-315H). Injectionports 315 introduce concentrate solutions 115 into static mixing chamber310. In particular, injection ports 315 introduce concentrate solutions115 into turbulent diluent stream 125. FIG. 6 shows a mechanical drawingof static mixing chamber 310 in further detail.

FIG. 6A, FIG. 6B, and FIG. 7 illustrate static mixing chamber 310 ingreater detail. In particular, FIGS. 6A and 6B are mechanical drawingsshowing a front view and a right side view, respectively, of staticmixing chamber 310. FIG. 7 is an isometric drawing of static mixingchamber 310. As shown in FIGS. 6A, 6B, and 7, static mixing chamber 310includes a series of injection ports 315. In particular, static mixingchamber 310 includes two groupings of radially disposed injection portsshown as injection ports 315C, 315D, and 315E and injection ports 315F,315G, and 315H. In addition, as shown in FIGS. 6A and 6B, static mixingchamber 310 also includes two additional injection ports 315A and 315B.

Injection ports 315C, 315D, and 315E are described as being radiallydisposed around static mixing chamber 310. By “radially disposed” it ismeant that injection ports 315C, 315D, and 315E are located on a commoncircumference around static mixing chamber 310. That is, injection ports315C, 315D, and 315E are located at an approximately equal distance fromthe upstream end of static mixing chamber 310. Preferably, injectionports 315C, 315D, and 315E are spaced equally about the commoncircumference of static mixing chamber 310. Thus, for the case of threeinjection ports, the injection ports 315C, 315D, and 315E are space at120 degree increments. Other embodiments may provide for non-equalspacings about the common circumference.

In one embodiment of the present invention, the injection ports areessentially disposed both “linearly” and “radially” from one another.Such would be the case, for example, where the injection ports weredisposed in spiral fashion about static mixing chamber 310. Depending onthe length of the spiral, the injection ports could be consideredlinearly disposed, radially disposed, or both.

Injection ports 315F, 315G, and 315H are also radially disposed aroundstatic mixing chamber 310. In addition, this group of injection ports,both individually and collectively, is “linearly disposed” along thefluid flow path of static mixing chamber 310 from injection ports 315C,315D, and 315E as shown in FIG. 6. In other words, injection ports 315F,315G, and 315H are located at an approximately equal distance from theupstream end of static mixing chamber 310, where this distance issufficiently different from the distance from the upstream end of staticmixing chamber 310 to injection ports 315C, 315D, and 315E.

In the particular embodiment shown in FIG. 6 and FIG. 7, three injectionports are radially disposed from one another in each of the two groupsof injection ports. As would be apparent to one skilled in the art,additional injection ports may be included within each group, limited bytwo parameters. The first parameter is the number of injection portsthat can physically, or mechanically, fit around static mixing chamber310. The second parameter is the number of injection ports that can beused to introduce concentrate solutions 115 to diluent 125 without theingredients of concentrate solutions 115 adversely chemically reactingwith one another. As also would be apparent, fewer injection ports maybe included within each group.

In addition to changing the number of injection ports within eachradially disposed group, the number of radially disposed groups may alsobe changed. The number of radially disposed groups of injection ports isalso limited by the same parameters as described above as would beapparent.

As shown in FIG. 6 and FIG. 7, diluent 125 flows from the upstream endof static mixing chamber 310 toward the downstream end of static mixingchamber 310. Thus, as diluent 125 flows through static mixing chamber310, diluent 125 encounters injection ports 315A and 315B first,followed by injection ports 315C, 315D and 315E, and finally, injectionports 315F, 315G and 315H.

As thus described, static mixing chamber 310 provides two manners inwhich different concentrate solutions 115 can be added to diluent 125.The first manner is to add the different concentrate solutions 15 byusing injection ports that are radially disposed from one another suchas injection ports 315F, 315G, 315H or injection ports 315C, 315D and315E. The second manner in which different concentrate solutions 115 canbe added to diluent 125 is by using injection ports 315 that arelinearly disposed from one another such as injection ports 315C and315F. In either case, an injection port 315 adds a concentrate solution115 to diluent 125 in a manner such that the concentrate solution 115becomes sufficiently diluted by diluent 125 prior to encountering anyother concentrate solution 115 added from a different injection port315. This prevents any adverse chemical reaction between the ingredientsof the two concentrate solutions.

While this is true in general, the order of introduction of certainconcentrate solutions 115 to diluent 125 from a particular injectionport configuration are preferred, while other orders of introduction arediscouraged. For example, medium product 135 that includes a basesoluble concentrate solution and a group II salts concentrate solutionare preferably prepared by introducing these two concentrate solutionsinto diluent 125 by radially disposed injection ports. Doing so improvesthe microenvironment chemistry of the resulting medium product 135.

Also, medium product 135 that includes a group II salts concentratesolution and an acid soluble concentrate solution are preferablyprepared by introducing these two concentrate solutions into diluent 125from linearly disposed injection ports 315. Introducing these twoconcentrate solutions from injection ports that are radially disposedfrom one another is detrimental to product quality and may create anirreversible precipitation of critical cell culture medium componentsrendering the resulting medium product inactive.

In a preferred embodiment of the present invention, the followinginjection ports 315 concentrate solution 115 pairings are used: acidsoluble concentrate solutions are introduced by injection port 315D;group I salts concentrate solutions are introduced by injection port315E; group II salts concentrate solutions are introduced by injectionport 315G; base soluble concentrate solutions are introduced byinjection port 315H; acid solutions for adjusting pH are introduced byinjection port 315A; and base (caustic) solutions for adjusting pH areintroduced by injection port 315B. If sodium hydroxide concentratesolutions are used, they are preferably introduced by injection port315F. Otherwise, injection port 315F is reserved for other concentratesolutions not included above. Injection port 315C is also reserved forother concentrate solutions not included above.

Medium Surge Vessel

FIG. 4 illustrates medium surge vessel 140 in greater detail. Mediumsurge vessel 140 includes a medium surge tank 410, an agitation system420, a level indicator 430, a temperature control system 450, and a pHsensor 470. Medium product 135 from medium mixing system 130 entersmedium surge tank 410 which provides a buffering mechanism for ALMS 100.In other words, medium surge vessel 140 provides a “buffer” between thecontinuous operation of medium mixing system 130 and the discontinuousoperation of downstream components of ALMS 100 such as fill system 170.Thus, medium product 135 from medium mixing system is permitted toaccumulate in medium surge vessel 140 when, for example, fill system 170is temporarily shutdown to change fill containers.

An amount of medium product 135 in medium surge tank 410 is monitored bycomputer control system 105 via fill indicator 430. Depending on thelevel of medium product 135 in medium surge tank 410, computer controlsystem 105 adjusts the output rate of medium product 135 from mediummixing system 130.

A pH level of medium product 135 is measured by pH sensor 470 as mediumproduct 135 leaves medium surge tank 410. This permits computer controlsystem 105 to monitor and ensure the quality of medium product 135.

In one embodiment of the present invention, agitation system 420 is usedto provide agitation (i.e., mixing) to medium product 135 within mediumsurge tank 410. In one embodiment, agitation system 420 providescontinuous mixing of medium product 135 in medium surge tank 410. Inanother embodiment, agitation system 420 provides mixing of mediumproduct in medium surge tank 410 after a particular level is reached orsome other parameter. Agitation system 420 may or may not be required inorder to maintain medium product 135 in a homogeneous state. In apreferred embodiment of the present invention, agitation system 420 isnot used.

In one embodiment of the present invention, temperature control system450 controls the temperature of medium product 135 within medium surgetank 410. Temperature control system 450 operates so as to maintain aparticular temperature of medium product 135 in medium surge tank 410.Various means of controlling the temperature of the contents of mediumsurge tank 410 are available as would be apparent. In one embodiment ofthe present invention, glycol is circulated through an outer tank (notshown) around medium surge tank 410 thereby maintaining a particulartemperature of the contents within medium surge tank 410. In a preferredembodiment of the present invention, temperature control system 450 isnot used.

In one embodiment of the present invention, compressed air 460 isprovided to medium surge tank 410 to maintain a given head pressurewithin medium surge tank 410. Compressed air 460 is used to providesufficient pressure to move medium product 135 through medium surge tankinto prefiltration system 150. In a preferred embodiment of the presentinvention, the head pressure is maintained between 6 and 10 p.s.i.g.Other embodiments may utilize gases other than air, such as nitrogen, toprovide the head pressure as well as to prevent the outgasing frommedium product 135 as would be apparent.

Diverter valve 445 is controlled by computer control system 105 toimplement the CIP operation as will be discussed below. Diverter valve445 diverts fluid to spray ball 440 in order to clean the inside ofmedium surge tank 410 during the CIP operation.

Filtration System

FIG. 5 illustrates prefiltration system 150 and sterile filtrationsystem 160 in further detail. Prefiltration system 150 includes aprefiltration pump 510 and a prefiltration filter 520. Prefiltrationsystem 150 receives medium product 145 from medium surge tank 140.Prefiltration pump 510 pumps medium product 145 through a non-sterileprefilter filter 520. Prefilter filter 520 is a filter membrane thatprovides variable filtration of medium product 145. Depending upon theparticular medium product 145 being prepared, the filter membrane isselected to filter particles that may range between 0.1 and 2 microns.

Medium product that has been filtered by prefiltration system 150 enterssterile filtration system 160. As shown in FIG. 5, sterile filtrationsystem 160 operates in a clean area 180. Sterile filtration system 160includes two sterilizing filters 530A and 530B in a parallelconfiguration followed by a final sterilizing filter 540. Thisparticular configuration of sterilizing filters provides redundant 0.1or 0.2 micron filtration for medium product 145. Filtered medium product165 is output from sterile filtration system 160 and enters fill system170.

Sterilizing filters 530 and final sterilizing filter 540 are steamsterilized via a steam in place operation which is discussed in furtherdetail below. In a preferred embodiment, the sterilizing filters aresteam sterilized prior to manufacturing a new batch of cell culturemedium formulation.

Fill System

As shown, fill system 170 is also contained within clean area 180. Fillsystem 170 provides aseptic connections in clean area 180 so thatmultiple medium product containers can be filled outside of clean area180.

In one embodiment of present invention, fill system 170 provides amechanism whereby multiple containers (i.e., sterile bags, carboys,glass bottles, drums, etc.) can be filled. In another embodiment of thepresent invention, fill system 170 may not be required or may bemodified. For example, an embodiment of ALMS 100 may be implemented toprovide medium product 145 directly to a bioreactor as would beapparent.

Diverter valves 505, 525 and 545 are controlled by computer controlsystem 105 and used during the CIP operation as will be discussed below.The diverter valves provide a mechanism to flush unwanted medium productthrough to waste disposal system 190 as well as to provide mechanisms toclean and product purge prefilter 520 and sterilizing filters 530A, 530Band 540.

ALMS Process Capability

In a preferred embodiment of the present invention, ALMS 100 is designedto operate with flow rates between 1,000 and 3,000 liters or mediumproduct per hour. Other embodiments of the present invention may havedifferent flow rates depending upon the sizing and accuracy of, forexample, concentrate solution pumps 340, diluent input pump 320, andstatic mixing chamber 310.

In a preferred embodiment of the present invention, medium product 165has an intra-run homogeneity with a precision tolerance of ±2.0%.Precision between production runs of medium product 165 from identicalconcentrated materials is ±3.0%. Furthermore, a pH fluctuation of mediumproduct 165 is within ±0.1 units.

Clean In Process (CIP) and Steam In Place (SIP) Process Operations

ALMS 100 is designed for on-line sanitization and sterilization in placeas required. The sanitization operation is commonly referred to as“clean in place.” The sterilization operation using steam under pressureis commonly referred to as “steam in place.” A typical operation willrequire sanitization of the entire system including WFI brake tank 230and steam sterilization of sterile filtration system 160 as well as fillsystem 170.

Sanitization of ALMS 100 includes the flushing of the entire ALMS 100with hot water from hot WFI 220. Hot water from hot WFI 220 is routedthrough ALMS 100 via diverter valves (e.g., diverter valve 145, divertervalve 505, diverter valve 525, diverter valve 545, etc.) to and throughspray balls (e.g., spray ball 240 and spray ball 440), and recirculatedfrom fill system 170 to media mixing system 130 via an appropriateconduit (shown as line 175 in FIG. 1) to flush ALMS 100. In oneembodiment of the present invention, caustic solution is added to hotwater from hot WFI 220 via static mixing chamber 310 to provide a hotcaustic sanitization of ALMS 100. The hot caustic is recirculated,neutralized with acid and sent to waste disposal system 190.

For sterilizing ALMS 100, steam is introduced at the sterile filtrationsystem 160 via a steam input port 550 located inside clean area 180.Steam flows through sterile filtration system 160, including sterilizingfilters 530 and final sterilizing filter 540, and fill system 170, andheats these components to sterilization temperatures. The temperature ismonitored at appropriate points and sterilization is confirmed usingwell known time/temperature parameters as would be apparent.

The by-products of the sanitization process are routed to waste disposalsystem 190 as shown in various figures. In one embodiment of the presentinvention, waste disposal system treats any by-products of ALMS 100 byappropriate measures so as not to introduce any harmful products intothe plant's waste disposal system as would be apparent.

Computer Control System

In various embodiments of the present invention, computer control system105 is implemented using hardware, software or a combination thereof andmay be implemented in a computer system or other processing system. Infact, in one embodiment, the invention is directed toward a computersystem capable of carrying out the functionality described herein. Anexample computer system 802 is shown in FIG. 8. Computer system 802includes one or more processors, such as processor 804. Processor 804 isconnected to a communication bus 806. Various software embodiments aredescribed in terms of this example computer system. After reading thisdescription, it will become apparent to a person skilled in the relevantart how to implement the invention using other computer systems and/orcomputer architectures.

Computer system 802 also includes a main memory 808, preferably randomaccess memory (RAM), and may also include a secondary memory 810.Secondary memory 810 may include, for example, a hard disk drive 812and/or a removable storage drive 814, representing a floppy disk drive,a magnetic tape drive, an optical disk drive, etc. Removable storagedrive 814 reads from and/or writes to a removable storage unit 818 in awell known manner. Removable storage unit 818, represents a floppy disk,magnetic tape, optical disk, etc. which is read by and written to byremovable storage drive 814. As will be appreciated, removable storageunit 818 includes a computer usable storage medium having stored thereincomputer software and/or data.

In alternative embodiments, secondary memory 810 may include othersimilar means for allowing computer programs or other instructions to beloaded into computer system 802. Such means can include, for example, aremovable storage unit 822 and an interface 820. Examples of such caninclude a program cartridge and cartridge interface (such as that foundin video game devices), a removable memory chip (such as an EPROM, orPROM) and associated socket, and other removable storage units 822 andinterfaces 820 which allow software and data to be transferred from theremovable storage unit 818 to computer system 802.

Computer system 802 can also include a communications interface 824.Communications interface 824 allows software and data to be transferredbetween computer system 802 and external devices. Examples ofcommunications interface 824 can include a modem, a network interface(such as an Ethernet card), a communications port, a PCMCIA slot andcard, etc. Software and data transferred via communications interface824 are in the form of signals which can be electronic, electromagnetic,optical or other signals capable of being received by communicationsinterface 824. Signals 826 are provided to communications interface viaa channel 828. Channel 828 carries signals 826 and can be implementedusing wire or cable, fiber optics, a phone line, a cellular phone link,an RF link and other communications channels.

In this document, the terms “computer program medium” and “computerusable medium” are used to generally refer to media such as removablestorage device 818, a hard disk installed in hard disk drive 812, andsignals 826. These computer program products are means for providingsoftware to computer system 802.

Computer programs (also called computer control logic) are stored inmain memory and/or secondary memory 810. Computer programs can also bereceived via communications interface 824. Such computer programs, whenexecuted, enable the computer system 802 to perform the features of thepresent invention as discussed herein. In particular, the computerprograms, when executed, enable processor 804 to perform the features ofthe present invention. Accordingly, such computer programs representcontrollers of the computer system 802.

In an embodiment where the invention is implement using software, thesoftware may be stored in a computer program product and loaded intocomputer system 802 using removable storage drive 814, hard drive 812 orcommunications interface 824. The control logic (software), whenexecuted by processor 804, causes processor 804 to perform the functionsof the invention as described herein.

In another embodiment, the invention is implemented primarily inhardware using, for example, hardware components such as applicationspecific integrated circuits (ASICs). Implementation of the hardwarestate machine so as to perform the functions described herein will beapparent to persons skilled in the relevant art(s).

In yet another embodiment, the invention is implemented using acombination of both hardware and software.

CONCLUSION

While the invention has been particularly shown and described withreference to several preferred embodiments thereof, it will beunderstood by those skilled in the art that various changes in form anddetails may be made therein without departing from the spirit and scopeof the invention as defined in the appended claims.

What is claimed is:
 1. A method for continuously mixing aqueouschemically incompatible concentrate solutions, comprising the steps of:providing a flow controlled turbulent diluent stream; adding a pluralityof chemically incompatible concentrate solutions to said turbulentdiluent stream in such a manner that none of the ingredients of theconcentrate solutions adversely chemically react with each other; andthereby forming a diluted solution of said concentrate solutions.
 2. Themethod of claim 1, wherein said adding step comprises the step of:adding a first of said chemically incompatible concentrate solutions tosaid turbulent diluent stream at a first point sufficiently distant froma second point at which a second of said chemically incompatibleconcentrate solutions is added to said turbulent diluent stream, toprevent the ingredients of said first and said second chemicallyincompatible concentrate solutions from adversely chemically reactingwith each other.
 3. The method of claim 2, wherein said step of addingsaid first and second solutions comprises the step of: adding said firstof said chemically incompatible concentrate solutions to said turbulentdiluent stream in a manner that is approximately radially disposed fromthe addition of said second of said chemically incompatible concentratesolutions to prevent the ingredients of said first and said secondchemically incompatible concentrate solutions from adversely chemicallyreacting with each other.
 4. The method of claim 2, wherein said step ofadding said first and second solutions comprises the step of: addingsaid first of said chemically incompatible concentrate solutions to saidturbulent diluent stream linearly upstream of the addition of saidsecond of chemically incompatible concentrate solutions to prevent theingredients of said first and said second chemically incompatibleconcentrate solutions from adversely chemically reacting with eachother.
 5. The method of claim 2, further comprising the step of:adjusting a pH level of said turbulent diluent stream.
 6. The method ofclaim 5, wherein said step of adjusting a pH level comprises the stepof: adding an acid solution to said turbulent diluent stream to reducesaid pH level.
 7. The method of claim 5, wherein said step of adjustinga pH level comprises the step of: adding a caustic solution to saidturbulent diluent stream to increase said pH level.
 8. The method ofclaim 5, wherein said step of adjusting a pH level comprises the stepsof: adding an acid solution to said turbulent diluent stream to decreasesaid pH level; and adding a caustic solution to said turbulent diluentstream to increase said pH level.
 9. The method of claim 5, wherein saidstep of adjusting a pH level comprises the step of: adjusting a pH levelof said turbulent diluent stream prior to said step of adding saidplurality of chemically incompatible concentrate solutions to saidturbulent diluent stream.
 10. A method for continuously mixing aqueouschemically incompatible concentrate solutions, comprising the steps of:providing a flow controlled turbulent diluent stream; adding at leasttwo of a plurality of chemically incompatible concentrate solutions tosaid turbulent diluent stream in such a manner that none of theingredients of said at least two of said plurality of chemicallyincompatible concentrate solutions adversely chemically react with eachother, wherein said plurality of chemically incompatible concentratesolutions include solutions from the set of: an acid soluble concentratesolution subgroup, a group I salt concentrate solution subgroup, a groupII salt concentrate solution subgroup, and a base soluble concentratesolution subgroup; and thereby forming a diluted solution of saidconcentrate solutions.
 11. A method for continuously mixing aqueouschemically incompatible concentrate solutions, comprising the steps of:providing a flow controlled turbulent diluent stream; adding a basesoluble concentrate solution to said turbulent diluent stream in amanner that is approximately radially disposed to the addition of agroup II salt concentrate solution so that none of the ingredients ofsaid base soluble solution and said group II salt concentrate solutionadversely chemically react with each other; and thereby forming adiluted solution of said base soluble and group II salt concentratesolutions.
 12. A method for continuously mixing aqueous chemicallyincompatible concentrate solutions, comprising the steps of: providing aflow controlled turbulent diluent stream; adding an acid solubleconcentrate solution to said turbulent diluent stream; adding a group IIsalt concentrate solution to said turbulent diluent stream at a positionlinearly disposed from the addition of said acid soluble concentratesolution in such a manner that none of the ingredients of said acidsoluble concentrate solution and said group II salt concentrate solutionadversely chemically react with each other; and thereby forming adiluted solution of said acid soluble and group II salt concentratesolutions.